Nucleic acid sequences have a wide variety of applications in the field of molecular biology. They are a valuable tool in many analytical and application techniques used in the field of molecular biology, health and medicine (gene therapy, diagnostics, recombinant protein expression), bioterrorism (agent detection and analysis), forensics, space science, and food science. Some examples of these techniques include genotyping microorganisms, DNA fingerprinting plants and animals, detecting pathogens and beneficial microorganisms in soils, water, plants and animals, forensic identification of biological samples and environmental samples contaminated with different biological entities. All these techniques are based on identifying a specific sequence of nucleic acid in either a biological sample, such as a microorganism, plant tissues or animal tissues, or in any environment capable of supporting life. Identifying target nucleic acid sequences directly in biological samples and in environmental samples has the advantages of speed, accuracy, high-throughput and a low limit of detection to picogram or femtogram quantities of nucleic acids. The target nucleic acid sequence, in order to be used as a diagnostic tool in such applications, should be free of contaminants that inhibit PCR and other downstream applications. These contaminants are often from the groups that include polyphenols, polysaccharides and humic substances.
The field of nucleic acid extraction and subsequent amplification of this DNA by polymerase chain reaction (PCR) has revolutionized the rapid analysis of genetic composition of several ecosystems. Methods and kits are available for isolating genomic DNA from a wide range of biological entities, and from the environment in which these living entities dwell. The polymerase chain reaction (PCR) is a very powerful and sensitive analytical technique with applications in many diverse fields, including molecular biology, clinical diagnosis, forensic analysis, and population genetics. However, the success rate in soil and plant genomic analysis has been relatively slow due to the poor quality of DNA isolated. In plant genomic DNA analysis, the DNA is invariably co-extracted with other plant components such as polyphenols and polysaccharides which inhibit PCR applications.
In the field of soil ecosystems, nucleic acid extraction methods suffer from compounded inefficiencies of DNA sorption to soil surfaces and co-extraction of enzymatic inhibitors from soils. Both the clay and organic fractions of soil affect DNA isolation and purification. Clay has a tendency to bind DNA adsorptively, whereas humic polymers found in the organic fraction tend to co-purify with DNA during the extraction procedure. The higher the montmorillonitic clay and organic matter content, the higher the buffering capacity of the soil system and also greater the amount of DNA adsorbed to the soil particles. Thus methods developed for a particular soil type with a clay:organic ratio may not work for any other soil type with different clay:organic ratio. It has been previously reported that phenol extraction of DNA contaminated with humic substances resulted in lowering the DNA recovery efficiency. Compost may have a variety of additional organic compounds that may co-purify with DNA and inhibit enzymatic manipulations of the DNA. An additional concern when isolating microbial DNA from compost is that plant material in various stages of decomposition may be present in significant concentrations in compost.
Studies of higher organisms such as fungi, plants and animals, direct nucleic acid isolations are still plagued with quality issues. In cyanobacteria, fungi, algae and plants, pigments and cell wall components such as chitins and polysaccharides will inhibit PCR. These cell types are rich in endo—and exonucleases and contain photosynthetic pigments, which can inhibit enzymatic reactions, especially reverse transcription and PCR.
The nature of the contaminants in crude nucleic acid preparations from soils and sediments and their interactions with DNA and RNA are not well understood. Most frequently these contaminants are considered to be humic and fulvic acids and a heterogeneous mixture of phenolic polymers and oligomers. Humic substances are formed when microbes degrade plant residues and are stabilized to degradation by covalent binding of their reactive sites to metal ions and clay minerals. Humic substances consist of polycyclic aromatics to which saccharides, peptides, and phenols are attached. The predominant types of humic substances in soils are humic acids (HA, molecular weight of 300 kDa and greater) and fulvic acids (FA, molecular weight of as low as 0.1 kDa). Humic acids are soluble in alkaline pH and precipitate with hydrochloric or sulphuric acids at pH 1.0 to 2.0, while fulvic acids remain in solution even at acidic pH (Stevenson, 1994). Most frequently, DNA extracts from soils showing brown coloration are indicative of contamination with humic like substances. These brown compounds cannot be easily removed from DNA extracts. Solvent extraction of crude DNA extracts with solvents such as phenol, diethyl ether, acetone, methanol and ethanol were not successful in removing the brown coloration, and the DNA was still discolored and resistant to digestion by restriction endonucleases. Some of these compounds also appear to co-migrate with DNA during CsCl-ethidium bromide isopycnic ultracentrifugation, resulting in light brown coloration of the recovered DNA. These observations imply an intimate association between the contaminants and DNA. While the nature of the association between contaminating compounds and DNA has not been elucidated, the reversible and irreversible binding of polyphenols, such as tannins, to proteins is well understood.
Direct extraction of total nucleic acid from soils or sediments usually results in co-extraction of other soil components, mainly humic acids or other humic substances, which negatively interfere with DNA transforming and detecting processes. It has been reported that these substances inhibit restriction endonucleases and Taq polymerase, the key enzyme of PCR, and decrease efficiencies in DNA-DNA hybridizations. Separation of humic substances from DNA usually involves time-consuming and tedious steps. To circumvent this, size-exclusion chromatography and the use of polyvinylpolypyrrolidone spin columns have been widely used. Size-exclusion chromatography includes the use of SEPHADEX G-200™ or MICROSPIN S-400 HR™, while water-insoluble PVPP and water-soluble polyvinylpyrrolidone (PVP) as humic acid-binding agents have also been reported.